Project 2: Modeling the Pathogenic Processes of Shigella in the Human Colonoid Model Shigella is one of the most important diarrheal pathogens among children in developing countries. Recent data from the Global Enteric Multicenter Study (GEMS) identified Shigella as the most important pathogen in children in the two older age groups (encompassing children 12-59 months of age). Infections by this pathogen result in severe outcomes, increasing antibiotic resistance is limiting therapeutic options and there is no licensed vaccine for Shigella. The study of this enteric pathogen and advancement of new or improved vaccines and therapeutics has suffered from a lack of preclinical models that reflect relevant human disease pathophysiology. The human enteroid/colonoid ?mini-gut? model presents a technological leap in human GI system modeling that will allow identification of highly relevant host-pathogen interactions that are expected to lead to the discovery of novel points of intervention for therapeutic options and vaccine development. We propose to use this innovative system to answer questions about pathogenic processes that have not yet been sufficiently explored, including mechanisms of entry and invasion, apical and basolateral surface contributions, interactions with host cell factors including mucus, and the role of bacterial virulence factors including Pic. The development of the multi-cellular colonoids grown as a monolayers on Transwell filters yields direct access to both apical and basolateral surfaces which express physiologic mucus, facilitates the engraftment of immune cells such as macrophages and dendritic cells and allows quantification of neutrophil transmigration. This project will define optimal parameters for Shigella-colonoid infection studies that will allow identification of critical pathogenic mechanisms, and host cell responses. Furthermore, this system will be developed to serve as a preclinical model for evaluation of live attenuated Shigella vaccine candidates to accelerate advancement of urgently needed intervention strategies.